Use of antitumor compound C11

Abstract

The application belongs to the field of medicine and relates to the application of an antitumor compound C11. Specifically, the application relates to the application of a compound C11 and crystal forms, pharmaceutically acceptable salts, esters, hydrates, solvates or prodrugs thereof in the preparation of NME4 protein inhibitors, wherein the structural formula of the compound C11 is shown as formula I. In vitro experimental results show that C11 has a good inhibitory effect on multiple cell lines of colorectal cancer, and the target of the effect is NME4; animal experiments show that C11 can effectively inhibit the growth of colorectal cancer in nude mice. The above results show that the compound C11 can be used as an inhibitor of NME4 and as a drug for tumors with high expression of NME4, in particular as a drug for the clinical treatment of colorectal cancer. The application provides a new therapeutic drug for colorectal cancer, provides a new way to overcome drug resistance of the disease, and provides a new treatment strategy for the clinical treatment of other types of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, in particular, to the new use of anti-tumor compound C11. BACKGROUND

[0002] Colorectal cancer is a digestive tract tumor found in all age groups. According to the global cancer research report, the global incidence of colorectal cancer ranks third, and the mortality rate ranks second. In the Chinese cancer research report, the incidence of colorectal cancer in China ranks second among all cancers, and the mortality rate ranks fourth. Colorectal cancer patients often show digestive tract bleeding, obstruction, etc. in clinical practice, and end-stage patients often die of multiple organ metastasis and failure. Unfortunately, there is no complete cure for colorectal cancer at present, so limiting the growth of colorectal cancer is still the focus of colorectal cancer drug development.

[0003] Nucleoside diphosphate kinase 4 (NME4) is located on the inner membrane of mitochondria and mainly participates in the mutual transformation of ATP / ADP and GTP / GDP through the transfer of high-energy phosphate groups. Previous research results suggest that NME4 is highly related to cystic fibrosis and Barth syndrome. However, current research suggests that NME4 is elevated in gastric cancer and colorectal cancer, but there is no relevant research on the pathogenesis of colorectal cancer. SUMMARY

[0004] The purpose of the present application is to provide a new use of compound C11.

[0005] Compound C11 (CAS No: 31980-12-8, also referred to as C11 herein) is recorded in the pubchem compound library, and the chemical structural formula is C9H 10 N2O2; molecular weight: English name 2,2-Dimethyl-2H-benzimidazole 1,3-dioxide. The chemical structural formula is shown in Figure I:

[0006]

[0007] Compound C11 was first synthesized by V.A. Samsonov and L.B. Volodarskii of Novosibirsk Organic Chemistry Association. The synthesis method is to react cyclohexanedione and cycloheptanedione 1,2-dioxime with acetone, cyclopentanone, and methyl ethyl ketone to obtain the corresponding 2H-imidazole 1,3-dioxide. There is no record of the use of compound C11 as a drug at present.

[0008] Through computer virtual screening, the applicant found that compound C11 binds to NME4 and can enter the active pocket. From this discovery, the present application is completed.

[0009] To achieve the above object, the first aspect of the present application provides the use of compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug in the preparation of NME4 protein inhibitor.

[0010] The second aspect of the present application provides the use of anti-tumor compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug in the preparation of NME4 high expression tumor cell proliferation inhibitor. The compound C11 targets NME4 protein and inhibits the activity of NME4.

[0011] According to a preferred embodiment of the present application, the NME4 high expression tumor cell is colorectal cancer cell.

[0012] The embodiments of the present application prove that compound C11 has killing activity on multiple cell lines of colorectal cancer, including but not limited to HCT116, SW620, DLD-1, MC38, CT26 or HROC374 cell lines.

[0013] The third aspect of the present application provides the use of anti-tumor compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug in the preparation of a drug or pharmaceutical composition for preventing and / or treating NME4 high expression tumor. Compound 11 targets NME4 gene or protein, inhibits tumor cell proliferation, and achieves the purpose of preventing and treating tumor.

[0014] According to a preferred embodiment of the present application, the NME4 high expression tumor is colorectal cancer.

[0015] The anti-tumor compound C11 and its crystal form, pharmaceutically acceptable salt, ester, hydrate, solvate or prodrug are used as active ingredients in the drug, and as one of the active ingredients in the pharmaceutical composition, which further contains at least one other drug. The other drug is a drug with synergistic anti-tumor effect, such as a drug for preventing and / or treating colorectal cancer.

[0016] In the present application, the "prodrug" refers to an agent that is converted into the prototype drug in vivo. The prodrug is usually a drug precursor, which is converted into an active substance by administration and absorption, or is changed into a more active species through some process, such as metabolic pathway. Some prodrugs have chemical groups that make them less active and / or have different solubility or some other properties compared to the prototype drug. Once the chemical group of the prodrug is removed and / or modified, the active drug is obtained.

[0017] In the present application, the drug and the pharmaceutical composition can be in solid or liquid form, and the dosage form can be tablets, dispersible tablets, lozenges, orally disintegrating tablets, sustained-release agents, capsules, soft capsules, dripping pills, granules, injections, powder injections or aerosols, etc. When the compound of the present application is used for the above-mentioned purposes, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, sustained-release agents, etc., and can be orally administered in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, 0.05-5% suspending agent), sugar syrups (containing, for example, about 10-50% sugar), and elixirs (containing 20-50% ethanol), or administered topically in the form of ointments, gels, drug-containing adhesive tapes, etc., or administered parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agent in an isotonic medium). Suitable administration routes include, but are not limited to, oral, intravenous injection, rectal, aerosol, parenteral administration, ocular administration, pulmonary administration, transdermal administration, vaginal administration, aural administration, nasal administration and topical drug.

[0018] The technical effect of the present application is that:

[0019] Based on the inhibitory activity of compound C11 on NME4, the present application provides a new anti-tumor use of compound C11. The in vitro experimental results show that C11 has good inhibitory effect on multiple cell lines of colorectal cancer, and the target is NME4; animal experiments show that C11 can effectively inhibit the growth of colorectal cancer in nude mice. The above results show that compound C11 can be used as an inhibitor of NME4, as a drug for tumors with high expression of NME4, and especially as a drug for the clinical treatment of colorectal cancer. The present application provides a new treatment drug for colorectal cancer, and provides a new way to overcome drug resistance of the disease, and also provides a new treatment strategy for the treatment of other types of tumors in clinical practice.

[0020] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0021] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

[0022] Figures 1-4 respectively, are the inhibition curves of compound C11 on colorectal cancer cells HCT116, SW620, DLD-1 and MC38.

[0023] Figures 5-9 is the animal experiment result of compound C11 inhibiting colorectal cancer.

[0024] Figure 10 is the binding result of compound C11 and NME4 drug target. Detailed Implementation

[0025] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0026] Unless otherwise specified in the examples, all procedures were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0027] Example 1 C11 compound killing activity on colorectal cancer cells

[0028] 1. Experimental materials:

[0029] (1) Cell lines: Human or mouse colorectal cancer cells (HCT116 cells, DLD-1 cells, SW620 cells, MC38 cells) were cultured in the corresponding culture medium containing 10% fetal bovine serum.

[0030] (2) Main reagents: RPMI 1640 medium (Gbico, USA), DMEM medium (Gbico, USA), C11 compound, Cell counting kit 8 kit (CCK8, Yisheng, China).

[0031] (3) Main instruments: carbon dioxide incubator (Thermo Forma, USA), fully automated microplate reader (Tecan, Switzerland).

[0032] 2. Experimental Methods:

[0033] HCT116, DLD-1, SW620, and MC38 cell lines were adherent cells cultured in appropriate medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCT116 and DLD-1 cells in logarithmic growth phase were seeded into sterile 96-well plates at a density of 1×10⁶ cells / well. 3 / well, 99μL of culture medium, 1μL added to each well to achieve final drug concentrations of 1, 5, 10, 25, and 50μM. Six replicates were set up for each sample, along with positive and blank controls. After 72 hours of incubation, CCK8 solution, 10μM / well, was added, and the mixture was incubated at 37℃ for 1 hour. The OD value was measured at 450nm using a microplate reader. The cell proliferation inhibition rate (IR%) was calculated using the following formula: = [(Control OD - Blank OD) - (Sample OD - Blank OD)] / (Control - Blank) × 100%

[0034] 3. Experimental Results

[0035] Figures 1-4The killing activity of compound C11 against HCT116, SW620, DLD-1, and MC38 cell lines was shown, with IC50 values ​​of 21.43 μM, 2.201 μM, 126.8 μM, and 5.679 μM, respectively. This demonstrates that compound C11 exhibits significant killing activity against multiple colorectal cancer cell lines in humans and mice.

[0036] Example 2: Animal experiment against colorectal cancer

[0037] 1. Experimental materials:

[0038] (1) Cell lines: Mouse colon cancer cell line CT26 and human primary colorectal cancer cell line HROC374 were cultured in RPMI 1640 medium and DMEM / F12 medium (Gbico, USA) containing 10% fetal bovine serum, respectively.

[0039] (2) Experimental animals: Female BALB / C mice (inoculated with CT26 cells) and female BALB / C nude mice (inoculated with HROC374 cells) were housed in an SPF-grade environment.

[0040] 2. Experimental Methods:

[0041] (1) Cell culture, same as in Example 1.

[0042] (2) Containing 3x10 5 CT26 cells and 2x10 6 HROC374 cells were injected subcutaneously into the left axilla of BALB / c mice and BALB / c nude mice, respectively. Once the tumors had grown and were measurable, the mice were randomly assigned to the control and treatment groups. Mice in the treatment group received daily intraperitoneal injections of compound C11 at 25 mg / kg and 50 mg / kg, respectively, while mice in the control group received an equal volume of solvent (2% DMSO + 40% PEG + 58% sterile water). Tumor size was measured every two days (length and width were measured; volume = 1 / 2 x length x width). 2 The ethical limit (2500m) will be reached once the first mouse is infected. 3 The mice were euthanized and photographed.

[0043] 3. Experimental Results

[0044] The results of CT26 cell inoculation are as follows Figures 5-7 As shown. Figure 5 The images show tumor images of mice in the drug-treated group and the control group after being inoculated with CT26 cells. It can be seen that the tumor volume of the two drug-treated groups was significantly reduced compared to the control group. Figure 6 The curve showing the change in tumor volume over time also confirms that the tumor volume in the treatment group mice was significantly different from that in the control group. Figure 7The tumor weights of each group are shown. It can be seen that the tumor weights of the control group mice are significantly higher than those of the drug administration groups, and there is no significant difference between the two drug administration groups.

[0045] The results of inoculating HROC374 cells are shown in Table 1. Figures 8-9 Figure 8 The tumor photos of the drug administration group mice and the control group mice inoculated with HROC374 cells are shown. It can be seen that, compared with the control group, the drug administration group mice also show a significant reduction in tumor volume. Figure 9 The tumor volume-time curve also confirms that the tumor volume of the drug administration group mice is significantly different from that of the control group.

[0046] The above animal experiments show that the C11 compound can effectively inhibit the growth of colorectal cancer in nude mice.

[0047] Example 3: C11 compound binding experiment with target NME4

[0048] 1. Experimental materials:

[0049] (1) Cell strain: human colon cancer cell line HCT116.

[0050] (2) Main reagents: 5X western loading buffer, phosphate buffered saline (PBS), mouse anti-NME4 monoclonal antibody (TA50110, China Aorun Dongyuan Company), mouse anti- ACTB monoclonal antibody (TA811000, China Aorun Dongyuan Company), horseradish peroxidase labeled goat anti-mouse IgG (H+L) (A0216, China Biyun Tian Company), protease inhibitor (P0016, China Biyun Tian Company).

[0051] (3) Main instruments: metal bath heater, liquid nitrogen, 37°C constant temperature water bath, electrophoresis instrument, electric shaker, exposure instrument (China Tanon Company)

[0052] 2. Experimental method: C11 compound (final concentration 25 μM) was added to the HCT116 cell culture dish for 6 hours, and an equal amount of DMSO solvent was used as a control. After washing with PBS, 1 mL of PBS containing protease inhibitor was added again, collected with a cell scraper and divided into 6 parts, each 100 μL. Set the temperature gradient: 42°C, 48°C, 55°C, 62°C, 67°C, 75°C, and each sample was placed in the corresponding temperature metal bath for heating for 3 minutes, and then the cells were lysed by liquid nitrogen-37°C cycle 3 times. After centrifugation at 20000g, 4°C for 10 minutes, the supernatant was collected. The supernatant sample was added with 1 / 4 sample volume of loading buffer and mixed well, and then heated in a 95°C metal bath for 10 min. The sample was then subjected to electrophoresis-membrane transfer-blocking-antibody incubation according to the standard western blot procedure, and finally the band was exposed and saved after exposure. ​

[0053] 3. Experimental results

[0054] The experimental results are shown in Figure 10 . Figure 10 The left panel is the WB electrophoresis graph, and the right panel is the graph based on the blackness of the bands of DMSO-NME4 group and C11-NME4 in the WB electrophoresis graph and the temperature.

[0055] It can be seen from Figure 10 that the protein content of NME4 of the C11 compound group is significantly higher than that of the DMSO group under the same temperature conditions, indicating that the C11 compound is combined with the NME4 protein and maintains its stability, so it is not easy to be degraded by high temperature.

[0056] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. Use of compound C11 and pharmaceutically acceptable salts thereof in the preparation of an inhibitor of proliferation of NME4 high expressing tumor cells, wherein the NME4 high expressing tumor cells are colorectal cancer cells, and wherein the compound C11 has a structural formula as shown in Formula I: Formula I.

2. The use according to claim 1, wherein, Formula I wherein the colorectal cancer cells are HCT116, SW620, DLD-1, MC38, CT26 or HROC374 cell lines.

3. Use of antitumor compound C11 and pharmaceutically acceptable salts thereof in the preparation of a medicament or a pharmaceutical composition for preventing and / or treating NME4 high expressing tumors, wherein the NME4 high expressing tumors are colorectal cancer.

4. Use according to claim 3, wherein, The medicament or the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers or excipients.

5. The use according to claim 3, wherein, The active ingredient of the medicament is the antitumor compound C11 and pharmaceutically acceptable salts thereof as claimed in claim 1.

6. The use according to claim 3, wherein, The active ingredient of the pharmaceutical composition comprises the antitumor compound C11 and pharmaceutically acceptable salts thereof, and at least one other drug.

7. The use according to claim 3, wherein, The dosage form of the medicament or the pharmaceutical composition is a tablet, a sustained release preparation, a capsule, a dripping pill, a granule, an injection, a powder injection or an aerosol.

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